miR-34a慢病毒表达质粒的构建及其对人结肠癌SW480细胞增殖的作用  

Construction of lenfiviral expression vector for miR-34a and its effect on proliferation of human colon cancer SW480 cells

作　　者：邵新宏[1] 张才全[1] 

机构地区：[1]重庆医科大学附属第一医院胃肠外科,重庆400016

出　　处：《中国生物制品学杂志》2013年第6期807-812,共6页Chinese Journal of Biologicals

摘　　要：目的构建miR-34a慢病毒表达质粒,并检测其对人结肠癌SW480细胞增殖的影响。方法以hsa-miR-34a前体序列pre-miR-34a为模板,设计末端部分互补的引物,进行引物退火反应,形成引物二聚体,进行PCR扩增,再以此DNA双链为模板进行PCR扩增,PCR产物酶切后,插入线性化的慢病毒载体pGIPZ-GFP中,构建重组慢病毒表达质粒pGIPZ-miR-34a,与包装质粒Helper 1.0、Helper 2.0共转染293T细胞,进行慢病毒的包装,梯度稀释法检测重组慢病毒的滴度。将包装好的重组慢病毒感染SW480细胞,qPCR法检测感染细胞中miR-34a基因mRNA的转录水平;MTT和平板克隆法检测感染细胞的增殖活力;流式细胞术检测感染细胞的细胞周期分布。结果 PCR及测序结果证实重组慢病毒表达质粒构建正确;重组慢病毒的滴度为6.52×108TU/ml;重组慢病毒感染SW480细胞后,显著上调了细胞中miR-34a基因mRNA的转录水平,明显抑制了细胞的增殖活力(P<0.01),G0-G1期细胞比例明显增加(P<0.01)。结论成功构建了miR-34a慢病毒表达质粒,慢病毒感染SW480细胞后,能显著提高miR-34a的内源性表达,并抑制细胞的增殖,为进一步研究miR-34a的功能及作用机制奠定了基础。Objective To construct a lentiviral expression vector for miR-34a and investigate its effect on proliferation of human colon cancer SW480 cells.Methods Partially complementary forward and reverse primers were designed based on has-pre-miR-34a sequence,of which dimmers were formed by annealing and amplified by PCR.The obtained double stranded DNA was used as a template for further amplification by PCR.The PCR product was digested with restriction endonuclease and insered into linearized lentiviral vector pGIPZ.The constructed recombinant plasmid pGIPZ-miR-34a was co-transfected to 293T cells with packaging plasmids Helper 1.0 and Helper 2.0 for packaging.The obtained recombinant lentivirus was determined by gradient dilution.SW480 cells were infected with recombinant lentivirus and determined for transcription level of miR-34a mRNA by qPCR,for proliferative activity by MTT and plate cloning method,and for distribution of cell cycle by flow cytometry.Results Restriction analysis and sequencing proved that recombinant lentiviral expression vector was constructed correctly.The titer of obtained recombinant lentivirus was 6.52 × 108 TU/ml.The recombinant lentivirus up-regulated the transcription level of miR-34a mRNA while inhibited the proliferative activity and increased the percentage at G0-G1 phases of infected SW480 cells（P ＆lt; 0.01）.Conclusion The lentiviral expression vector for miR-34a was successfully constructed,which increased the endogenous expression of miR-34a and inhibited the proliferation of SW480 cells.It laid a foundation of further study on function and action mechanism of miR-34a.

关 键 词：微RNAS MIR-34A 慢病毒 结肠癌 细胞增殖 

分 类 号：Q782[生物学—分子生物学] R735.35[医药卫生—肿瘤]